The long-term goal of this project is to determine the role of proteolysis in normal lens development and in cataractogenesis. The work focuses on the activity of the ubiquitin/proteasome proteolytic pathway. The long-lived ocular lens cells, which lose the ability to synthesize proteins, must carry out the numerous essential reactions of proteolytic enzymes and at the same time prevent irreversible damage by these enzymes. A balance must be maintained, and studies are proposed to determine the means of regulating activity to keep the balance. If we understand the mechanism of control, we can determine if a failure of the mechanism is related to cataractogenesis. Specific aim I is to complete studies on expression of ubiquitin-, proteasome proteolytic pathway components in rat lens epithelial cell explants to test the hypothesis that both 20S and 26S forms of the proteasome play a role in lens differentiation. Competitive RT-PCR assays will be used to quantitate mRNA in explants induced to differentiate by culturing in the presence of bFGF. Inhibitor studies will directly test for a role for the proteasome in differentiation. Specific aim II is to determine the importance of the 20S form of the proteasome in degradation of oxidatively modified proteins. A lens epithelial cell line, HLE B-3 cells, will be treated with hydrogen peroxide to model oxidative stress. The role of the 20S proteasome and ubiquitin in degradation of specific oxidatively modified proteins will be determined using proteasome inhibitors and co-immunoprecipitation analysis. Specific aim III will define the interaction between the proteasome and alpha-crystallin, which (1) is a substrate of the proteasome, (2) can function as a chaperone at high temperature, and (3) inhibits some activities and (4) activates others. The isolated subunits of alpha-crystallin have markedly different interactions with the proteasome. Purified and recombinant alpha-crystallin subunit interaction with the proteasome will be studied in enzyme assays with peptide and protein substrates.